Optimal timing for processing and cryopreservation of umbilical cord haematopoietic stem cells for clinical transplantation

Transcription

1 Bone Marrow Transplantation, (1999) 23, Stockton Press All rights reserved /99 $12. Optimal timing for processing and cryopreservation of umbilical cord haematopoietic stem cells for clinical transplantation AA Shlebak, SB Marley, IAG Roberts, RJ Davidson, JM Goldman and MY Gordon Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London, UK Summary: Some of the factors that may influence the number and quality of cord blood haematopoietic progenitor cells available for transplantation have been investigated including site of collection, delayed processing after collection and cryopreservation protocol. We used the granulocyte macrophage progenitor (CFU-GM) and erythroid burst-forming unit (BFU-E) assays to quantify progenitors. The capacity of CFU-GM to produce secondary colonies was used as a measure of progenitor cell quality. We found that: (1) there were no significant differences in total nucleated cells (TNC), mononuclear cells (MNC), CFU-GM or BFU-E numbers in paired specimens from the umbilical vein or veins at the base of the placenta. The potential of the CFU-GM to produce secondary colonies from the two sites was similar; (2) storing cord blood at room temperature or at 4 C resulted in a significant reduction in progenitor cell numbers beyond 9 h; and (3) cryopreservation following either controlled rate freezing or passive cooling reduced MNC numbers, viability and CFU-GM survival insignificantly but the potential of CFU-GM to produce secondary colonies was significantly reduced post cryopreservation (P.4). We conclude that the yield of CB progenitor cells is not affected by the site of collection, but is adversely affected by delays between collection and cryopreservation. Furthermore, cryopreservation reduced the CFU-GM potential to produce secondary colonies. Measures of progenitor cell quality as well as quantity may be relevant to assessing CB blood collections. Keywords: umbilical cord blood; timing of processing; cryopreservation The potential role of cord blood (CB) as a source of haematopoietic stem cells for use in transplantation was reviewed by Broxmeyer et al. 1 The first transplant using an HLAidentical sibling was performed in a Fanconi anaemia patient in Since then, many CB banks have been established 3,4 and over 5 CB transplants using both sibling and unrelated donors have been undertaken world-wide Correspondence: Prof MY Gordon, Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London W12 NN, UK Received 8 June 1998; accepted 2 September 1998 for the treatment of various malignant and non-malignant diseases. 5 1 However, many practical issues remain unresolved. Whereas sufficient stem cells for rapid and safe engraftment can routinely be harvested and stored from marrow and mobilised peripheral blood, the availability of stem cells in CB is limited. 11 This is a particular problem for adult recipients who require a higher total cell dose for ultimate engraftment. Indeed, recent data show that the clinical outcome of CB transplants depends on the number of nucleated cells/kg infused. 5 However, the minimum dose required for safe CB engraftment is not yet known. 12 Several methods for collection of CB have been described. Broxmeyer et al 13 described a two-phase method for collection of cord blood. In the first step blood is collected from the severed cord during placental delivery; in the second phase additional blood was collected from multiple needle aspirations of placental veins. Turner et al 14 modified the above method in an effort to overcome placental venous collapse, reduce the risk of inadvertent needle stick injury and improve stem cell yield. Significantly larger volumes ( ml) were collected compared to 1 15 ml previously reported. Whole cord blood donations have been transplanted successfully 1,5,7 and, since early attempts at separating cord blood by density gradient techniques led to loss of MNCs, it was suggested that cord blood should be stored unseparated. 1,3 However, to optimise the use of space and to be cost-effective, cord blood needs to be stored as a separated product. In previous studies processing was universally completed within h after collection, with prior storage at 4 C or C 15,16 when necessary. While the viability and yield of stem cells after storage at 4 C has been reported to be reduced, storage at C is claimed to have no detrimental effects. 16 In this study we investigated the influence of site of collection, delay in processing of CB kept at 4 C or C and the effects of cryopreservation on the survival and quality of CB progenitor cells. Clonogenic assays have been used widely to evaluate haematopoietic progenitor cell in order to estimate the dose of stem cells available for haematological transplantation and to predict engraftment. 17,18 Here, we have used an additional parameter to measure the function of the progenitor cells by replating individual CFU- GM colonies into 96-well plates and observing secondary colony formation. This provides a measure of the ability of the CFU-GM to amplify in number and consequently relates to their ability to produce mature neutrophils.

2 132 Materials and methods CB collection and separation CB samples were collected into 2 ml sterile plastic universal containers (Sterilin, Stone, UK) containing preservativefree heparin (2 IU). This is the standard anticoagulant in use in this laboratory for collecting cells from peripheral blood and bone marrow for research although citrate phosphate dextrose is the standard anticoagulant for CB collection elsewhere. Cord blood was collected from the umbilical vein and/or veins at the base of the placenta after normal full-term deliveries, by a designated research midwife at Queen Charlotte s and Hammersmith Hospitals in London, UK. The approval of the local research ethics committee and informed consent from all pregnant mothers were obtained prior to collection. All aspects of cord clamping and placental delivery were at the midwives discretion, based on standard local practice. Total nucleated cell count (TNC) was estimated by haemocytometry after red cell lysis using 3% acetic acid. The CB sample was diluted 1:1 with Hank s balanced salt solution (HBSS) and the mononuclear cell (MNC) fraction was obtained by layering the sample on to an equal volume of Lymphoprep (Nycomed, Oslo, Norway) followed by centrifugation according to the manufacturer s instructions. The MNC were washed thrice (Gibco, Paisley, UK), and resuspended in -medium with the addition of 15% fetal calf serum (Gibco) before final MNC counts were obtained. Processing was completed within 2 4 h of collection, unless stated otherwise. Cryopreservation Separated MNCs were frozen using 1% dimethyl-sulphoxide (DMSO) with autologous serum. Samples of 2 ml containing 1 7 cells per tube were frozen using either the passive cooling method or controlled-rate freezing. For passive cooling the tubes were kept overnight at 7 C (Denley, Ultrafreezer, Billingshurst, UK), in tubs immersed in isopropranolol, and subsequently placed in the liquid phase of liquid nitrogen. Alternatively, the computerised controlled Table 1 Characteristics of samples derived from umbilical vein and placental veins (n 9) (t-test) Results Cord blood Placenta median (range) median (range) [P value] TNC 1 7 /ml.9 (.3 1.3).9 (.3 1.6) [.7] MNC 1 7 /ml.6 (.1 1.).5 (.1 1.) [.2] Day 7 CFU-GM 1.3 ( 2.5) 1.5 (.2 2.3) 1 3 /ml [.5] Day 14 CFU-GM.9 ( 1.1).8 (.2.9) 1 3 /ml [.1] BFU-E 1 3 /ml 1.1 (.4 1.9).9 (.1 1.2) [.3] Secondary GM 8.9 ( ) 8.2 (5 19) [.8] AUC 85.2 (56 136) 79 (74 112) [.9] rate freezer (Planer Biomed, Kryo 1, Series III, Sunbury, UK) was used according to the protocols established for processing stem cells for clinical transplantation. Initially, the sample s ambient temperature was lowered to 4 C for 5 min, followed by 1 C reduction per minute to 3 C then by 2 C reduction per minute to 6 C and finally by 4 C per minute to 1 C and held at 1 C until the sample was removed and placed in the liquid phase of liquid nitrogen. Cryopreserved MNCs were thawed rapidly in a waterbath (4 C), subsequently washed twice in a solution containing HBSS with the addition of 15% fetal calf serum (Gibco), heparin (1 U/ml) and DNAse 1 (1 U/ml). TNC and MNC numbers were counted. Viability was estimated by trypan blue exclusion. Clonogenic assays CFU-GM: Mononuclear cells (2 1 6 /ml) were incubated for 2 h in a tissue culture flask (Nunc, Naperville, IL, USA) at 37 C to remove adherent cells. The adherent celldepleted MNCs were then added to 3 ml of thawed methylcellulose and 3 l of a mixture of cytokines (all from First Link, West Midlands, UK), giving final concentrations of 1 5 MNC/ml and 2 ng/ml stem cell factor (SCF), 1 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF), 5 ng/ml interleukin-3 (IL-3), and 1 ng/ml granulocyte colony-stimulating factor (G-CSF). Following thorough mixing with a 1 ml syringe, the cells were plated into triplicate 35 mm diameter petri dishes, and incubated at 37 C in humidified CO 2 in air. Colonies, defined as aggregates of more than 5 cells and 2 cells were scored on days 7 and 14, respectively. On day 7 of incubation of primary cultures, 6 sequential colonies consisting of more than 5 cells were picked individually from the methylcellulose using an Eppendorf pipette. Each colony was gently transferred into a separate well of a 96-well microtitre plate containing 1 l of methylcellulose plus serum and cytokines. The colonies were dispersed to a single cell suspension and thoroughly mixed with the methylcellulose. Then the microtitre plates were incubated for a further 7 days at 37 C in humidified 5% CO 2 in air. Each well was scored for the presence and number of secondary CFU-GM consisting of more than 5 cells. BFU-E: Adherent cell-depleted MNC were adjusted to 1 6 /ml, added to a complete methylcellulose medium for colony assay of human cells (Methocult H4431; Metachem Diagnostics Ltd, Northampton, UK) at a concentration of 1 5 cells per ml and mixed thoroughly. Using a 1 l pipette the mixture was gently plated into flat-bottomed 96- well microtitre plates in aliquots of 1 l/well to facilitate colony scoring. Excess wells were filled with sterile water to reduce evaporative losses, incubated for 14 days then scored for BFU-E numbers. Analysis of data The data from the replating experiments were analysed using a Microsoft Excel spreadsheet version 5. (Microsoft Corporation, Redmond, WA, USA) on a Macintosh computer. The raw data are the number of the primary CFU-

3 GM producing secondary CFU-GM in numbers ranging from one to the maximum observed. A cumulative distribution was obtained for the proportion of primary CFU- GM producing more than n secondary CFU-GM. This proportion is plotted on a logarithmic scale (y axis) so that changes by the same proportion are given the same magnitude on the graph. The x axis is plotted as log 2 n to represent the number of cell divisions required to produce the observed number of secondary CFU-GM. Areas-under-thecurves (AUC) were calculated using the Trapezium rule. Non-parametric statistical analysis was performed using StatView SE software for the Macintosh computer (Abacus Concepts Inc, Berkeley, CA, USA). Results Site of sampling The influence of sampling site was investigated in paired samples (n 9) of equal volumes (2 ml) collected simultaneously from the umbilical vein and veins at the placental base. The two sets were then analysed in parallel for TNC, MNC, CFU-GM and BFU-E. No significant differences were found (Table 1). The potential of CFU-GM to produce secondary colonies on replating was also evaluated (n 5) to assess the progenitor cell quality and again the difference was statistically insignificant (P.89). Effects of storage time and time of processing The median time of arrival of CB specimens in the laboratory was 6 min after collection. The samples (n 1) were aliquoted into five equal volumes (6 1 ml), kept at C in the containers in which they were originally collected, and subsequently analysed following Ficoll separation immediately, after 6, 9, 12 and 24 h. There was a statistically significant reduction in day 7 CFU-GM beyond 9 h storage: median number (range) 1.9 (.5 2.7), 2. (.4 2.6),.78 (.3 1.2),.4 (.1.7) and /ml (.9.8) at, 6, 9, 12 and 24 h, respectively (Table 2). The recovery in terms of percentages was 41%, 21% and 23% at 9, 12 and 24 h, respectively. Day 14 CFU-GM were significantly reduced from 6 h onwards (Figure 1). The MNCs were not as greatly reduced, the percentage recoveries being 88%, 88%, 85% and 51% at 6, 9, 12 and 24 h, implying selective loss of progenitor/stem cells following prolonged storage. The effects of storing CB (n 7) samples at 4 C was investigated at two time points, and 9 h. For this, aliquoted equal volumes (1 ml) were analysed following Ficoll separation. A similar trend was observed with a significant reduction of day 7 CFU-GM (2.3 vs /ml) and day 14 CFU-GM (1.2 vs /ml) after 9 h. The percentage recovery of MNCs was 67% at 9 h (Figure 2). Effects of cryopreservation First, we compared computerised controlled rate freezing and freezing with passive cooling on paired CB samples (n 5). The median percentage of MNC recovery, viability and CFU-GM survival were 97 vs 9 (P.7), 93 vs 91 and 74 vs 78, respectively. The differences were insignificant (Table 3). We then explored the effect of cryopreservation on the quality of the CB CFU-GM as measured by their ability to produce secondary colonies after replating (n 5). This parameter was reduced post cryopreservation to a median (range) 14 (43 162) vs 136 (56 246) in the unfrozen controls. Although, the reduction in the AUC was modest, it was statistically significant (P.4) (Wilcoxon sign-rank test). Discussion In this study we investigated the influence of several variables on the quality and quantity of CB progenitor cells. Firstly, regarding the site of collection there were no differences of statistical significance between the cord and placenta either in progenitor cell content or in the potential to 133 Table 2 Effects of storage of CB at C and 4 C on MNC and CFU-GM recoveries Time Storage at C Storage at 4 C (h) (n 1) (n 7) MNC Day 7 Day 14 MNC Day 7 Day /ml CFU-GM CFU-GM 1 7 /ml CFU-GM CFU-GM [%] 1 3 /ml 1 3 /ml [%] 1 3 /ml 1 3 /ml Median [%] [%] Median [%] [%] (range) Median Median (range) Median Median (range) (range) (range) (range) (.1.9) (.5 2.7) (.7 1.3) (.66 1.) (.4 3.4) (.1 1.9) 6.72 [88] 2. [15].45 [45] ND ND ND (.1.9) (.4 2.6) (.1.76) 9.72 [88].78 [41].3 [3].58 [67].74 [32].62 [54] (.1.9) (.3 1.2) (.1.54) (.44.76) (.2 1.6) (.8.9) 12.7 [85].4 [21].14 [14] ND ND ND (.1.88) (.1.7) (.5.3) [51].44 [23].5 [5] ND ND ND (.1.68) (.9.8) (.1.2) ND not done.

4 134 a b Percentage recovery Percentage recovery c Percentage recovery Figure 1 Effects of storage at C on CB; (a) % MNC recovery, on days (b) 7 and (c) 14 % CFU-GM formation. generate secondary colonies. Whatever method is employed to collect cord blood, the placenta only contains a finite amount of blood. It has been suggested that early clamping of the cord is associated with a 2 to 4-fold increase in the volumes collected 13,19 but this raises major ethical and Table 3 Comparison of two methods of cryopreservation (n 5) Results Passive cooling Controlled rate median (range) freezing median (range) [P value] % MNC recovery 9 (88 1) 97 (92 1) [.7] a % MNC viability 91 (7 93) 93 (9 95) [.14] % CFU-GM 78 (47 86) 74 (6 1) survival [.5] a Wilcoxon sign-rank test. medico-legal concerns, as it may deprive the infant of blood. 19,2 According to our data, it is important to process and cryopreserve CB quickly after collection. There was a significant reduction in all parameters beyond 9 h storage at room temperature or 4 C. Campos et al 16 found similar results on storing CB at 4 C prior to separation and freezing. Our results support the need for prompt processing and freezing of separated specimens preferably within 6 h. This is an important issue with possible major repercussions on routine clinical high-scale CB banking practices. As a significant proportion of deliveries occurs out of routine hours, it would require stem cell laboratories to operate round the clock with manpower and financial implications. Alternatively, deliveries could be selected so timely processing can be accomplished. Cryopreservation of processed MNCs and of CD34 fractionated stem cells has been extensively studied. Broxmeyer et al studied the effects of computer controlled freezing on the progenitor cell numbers and concluded there was insignificant loss of progenitor/stem cells with over 9% CFU-GM recoveries following prolonged periods of freezing. Others concluded that cryopreservation affects neither recovery nor the clonogenic capacity of haematopoietic progenitor cells 21 in unseparated frozen CB for up to 7 years. 14,18 Almici et al 22 reported 82% CFU-GEMM, 94% BFU-E, 82% CFU-GM, and 9% recovery for more primitive progenitor cells as tested in LTC-IC. In this study we analysed the effects initially of passive cooling on the MNCs numbers and potential of secondary replating. CB samples (n = 9) were analysed for CFU-GM and their replating ability before and after freezing. MNC numbers and viabilities were similar to those previously reported 16,21 with CFU-GM survival at 74%. Migliaccio et al 23 found an association between the dose of colony-forming cells (CFC) infused per kg and the time to absolute neutrophil count (ANC) 5/ l following CB transplantation. At least 8% of the patients receiving more than CFC/kg engrafted. Studying the qualitative aspects of thawed CB cells before infusing them into recipients may clarify the reasons for the lack of engraftment in the one-fifth of the recipients of the same CFC dose. One unit of CB in the same study contained CFC count below the detectable level ( 2.5 CFC/ l) but did engraft albeit with a delay. Therefore, qualitative assessment of CB haemato-

5 a Percentage recovery Percentage recovery Percentage recovery b c poietic cells post cryopreservation may provide important data that may influence the speed of engraftment. Additionally, the replating ability as measured by AUC was significantly reduced (P.4) indicating inferior function of the progenitor cells compared to fresh cells. Furthermore, direct comparison of the former method with the computerised controlled rate freezing produced no differences in the MNC viability and CFU-GM recoveries. Therefore, both methods can be used for CB banking. The choice would depend on local resources and preferences. The residual function of the progenitor/stem cells following cryopreservation is satisfactory as indicated by the results of clinical cord transplantation using CB cryopreserved for several years. However, it may become relevant in combination with other factors such as marginal volume or storage for prolonged periods before cryopreservation. In summary, progenitor/stem cells derived from umbilical vein and placental veins have identical constituents both in terms of quantity and replating potential. Processing of collected CB should optimally be undertaken within 6 h and definitely before 9 h. This seems to be the time window where least loss of MNCs and clonogenic progenitors, and presumably of stem cells, occurs. This is an important issue with possible major implications for routine CB banking. Cryopreservation produced a significant reduction on CB ability to produce secondary colonies on replating. Furthermore, there is no difference between the effects of passive cooling and computerised controlled rate freezing on CB yield. The combined undesirable effects of obtaining CB of low volumes, delayed processing and functional deterioration post cryopreservation may help to explain the failure of engraftment following CB transplants in some patients. Documentation of these factors in relation to the ultimate outcome of transplantation is required before this information may be suggested as a means for selecting cord blood samples with the most favourable properties. Acknowledgements This study was supported by The Leukaemia Research Fund of Great Britain and a grant from the Locally Organised Grant Scheme. We are grateful to Richard M Syzdlo for statistical advice. We are indebted to Trudy A Stevens in the Centre for Midwifery Practice at Queen Charlotte s Hospital for co-ordinating cord blood collection and dispatch. References Figure 2 Effects of storage at 4 C on CB; (a) % MNC recovery, on days (b) 7 and (c) 14 % CFU-GM formation. 1 Broxmeyer HE, Douglas GW, Hangoc G et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 1989; 86: Gluckman E, Broxmeyer HE, Auerbach AD et al. Haematopoietic reconstitution in a patient with Fanconi anaemia by means of umbilical cord blood from an HLA identical sibling. New Engl J Med 1989; 321: Rubinstein P, Rosenfield RE, Adamson JW, Stevens CE. Stored placental blood for unrelated bone marrow reconstitution. Blood 1993; 81: Gluckman E, Wagner J, Hows J et al. Cord blood banking for

6 136 haematopoietic stem cell transplantation: an international cord blood transplant registry. Bone Marrow Transplant 1993; 11: Gluckman E, Rocha V, Boyer-Chammard A. Outcome of cord blood transplantation from related and unrelated donors. New Engl J Med 1997; 337: Kurtzberg J, Laughlin M, Graham ML. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. New Engl J Med 1996; 335: Wagner JE, Kernan NA, Steinbruch M et al. Allogeneic sibling umbilical cord blood transplantation in children with malignant and non-malignant diseases. Lancet 1995; 346: Rubinstein P, Carnier C, Adamson J et al. New York Blood Centre s program for unrelated placental/umbilical cord blood transplantation: 243 transplants in the first three years. Blood 1996; 88: 142a (Abstr.). 9 Almici C, Carlo-Stella C, Wagner JE, Rizzoli V. Umbilical cord blood as a source of haematopoietic stem cells: from research to clinical application. Haematologica 1995; 8: Gluckman E, Thierry D, Lesage S et al. Clinical application of stem cell transfusion from cord blood. Transfus Sci 1992; 13: Fritsch G, Stimpfl M, Buchinger P et al. Does cord blood contain enough progenitor cells for transplantation? Hematother 1994; 3: Wagner JE, Broxmeyer HE, Byrd RL et al. Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment. Blood 1992; 79: Broxmeyer HE, Kurtzberg J, Gluckman E et al. Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation. Blood Cells 199; 17: Turner CW, Luzins J, Hutcheson C. A modified harvest technique for cord blood haematopoietic stem cells. Bone Marrow Transplant 1992; 1: Ademokun JA, Chapman C, Dunn J et al. Umbilical cord blood collection and separation for haematopoietic progenitor cell banking. Bone Marrow Transplant 1997; 19: Campos L, Roubin N, Guyotat D. Definition of optimal conditions for collection and cryopreservation of umbilical cord hematopoietic cells. Cryobiology 1995; 32: Douay l, Gorin NC, Mary JY et al. Recovery of CFU-GM from cryopreserved marrow and in vivo evaluation after autologous bone marrow transplantation are predictive of engraftment. Exp Hematol 1986; 14: Emminger W, Emminger-Schmidmeir W, Hocker P et al. Myeloid progenitor cells (CFU-GM) predict engraftment kinetics in autologous transplantation in children. Bone Marrow Transplant 1989; 4: Ende N. Cord blood collection: effects on newborns (medicolegal). Blood 1995; 86: Moss AJ, Monset-Couchard M. Placental transfusion: early versus late, clamping of the umbilical cord. Pediatrics 1967; 4: Harris DT, Schumacher MJ, Rychlik S. Collection, separation and cryopreservation of umbilical cord blood for use in transplantation. Bone Marrow Transplant 1994; 13: Almici C, Carlo-Stella C, Mangoni L. Density separation and cryopreservation of umbilical cord blood cells: evaluation of recovery in short- and long-term cultures. Acta Haematol 1996; 95: Migliaccio AR, Adamson JW, Rubinstein P, Stevens C. Correlation between progenitor cell dose, likelihood to engraft and time to myeloid engraftment in 13 unrelated placental/cord blood transplants. 3rd Eurocord Concerted Action Workshop, Annecy, France, 1998 (Abstr.).